Abstract 841: The role of C/EBPβ in prostate cancer cell survival.

Abstract The C/EBP family of transcription factors regulates cellular differentiation, proliferation, and inflammatory responses. Three isoforms of C/EBPβ are expressed from the same mRNA: the long LAP1 and LAP2 isoforms mediate transcriptional activation whereas the truncated LIP isoform is repressive. C/EBPβ is dysregulated in various malignancies including breast cancer or multiple myeloma however, its role in prostate cancer is not clear. We have recently reported that C/EBP proteins interact directly with DNA bound NF-kB p50 to displace HDAC, thereby de-repressing anti-apoptotic NF-kB target genes (Paz-Priel, et al. 2011). In four AR-positive (LNCaP, LAPC-4, C4-2B and CwR22Rv1) and two AR-negative (PC3, DU-145) cell lines Western blot analysis demonstrates presence of full-length LAP and the N-terminally truncated LIP isoforms of C/EBPβ. DU-145 cells were stably transduced with a panel of 5 C/EBPβ shRNAs (Open Biosystems). Two shRNA effectively reduced C/EBPβ RNA and protein levels and expression of BCL2 and MCL1 RNA and protein were diminished in parallel. Accordingly, DU-145 cells with reduced C/EBPβ showed increased apoptosis in the presence of paclitaxel compared to cells expressing a non targeting shRNA. We designed and generated a dominant-negative C/EBP termed A-C/EBP, containing 21 predominantly acidic amino acids linked to the C/EBPβ leucine zipper in place of the DNA binding basic region. The acidic domain in A-C/EBP mimics DNA, allowing A-C/EBP to hetero-dimerize with C/EBP proteins to interfere with their ability to bind DNA. Indeed co-transfection of A-C/EBP abrogated C/EBP activation of a Bcl2 promoter luciferase reporter. DU-145 or LNCaP cells expressing A-C/EBP or GFP control were exposed to paclitaxel and apoptosis was assessed after 48 hrs. Cells expressing A-CEBP exhibited increased annexin V positivity (6.0% vs. 15.2%, or 6.7% vs. 17.0% for DU-145 or LNCaP, respectively cultured with 3 nM of paclitaxel), indicating that blockage of C/EBP function or reduced C/EBPβ expression is synergistic with chemotherapy. DU-145 cells expressing A-C/EBP regulated by a TET-ON system, or GFP expressing control cells, were subjected to clonogenic assay in the presence or absence of doxycycline. On average, induction of A-C/EBP in DU-145 cells significantly lowered the number of colonies obtained compared with cells cultured without doxycycline (21.3 vs. 39.7 colonies per 100 seeded cell, p<0.02). Control cells, cultured - or + doxycycline formed 40.3 vs. 50.2 colonies per 100 seeded cell. Together our data suggest that C/EBPβ modulates the survival of both androgen-dependent and -independent prostate cancer cells, and that C/EBPβ inhibition may sensitize cells to chemotherapy. Citation Format: David Baraket, Jing Zhang, Theresa Barberi, Alan D. Friedman, Ido Paz-Priel. The role of C/EBPβ in prostate cancer cell survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 841. doi:10.1158/1538-7445.AM2013-841